Preparation of sterol and stanol-esters

ABSTRACT

The present invention provides a method for the direct esterification of stanols and sterols with fatty acids to form stanol/sterol-esters. The method provides a synthetic route that is amenable to large scale production of the esters in high yields. A preferred embodiment employs a food grade process free of organic solvents or mineral acids.

FIELD OF THE INVENTION

This invention relates to the preparation of discreet sterol andstanol-esters through a highly efficient acid catalyzed route.

BACKGROUND OF THE INVENTION

It has been shown that the addition of plant sterols, such as.β-sitosterol, to diets will reduce serum cholesterol levels. The sterolsreduce serum cholesterol through the disruption of intestinal absorptionof dietary cholesterol by displacing it from bile acid micelli. Morerecently, β-sitosterol's saturated derivative, β-sitostanol, has beenshown to be more effective in the reduction of intestinal cholesterolabsorption. The sitostanol itself is virtually unabsorbed, so it doesnot contribute at all to in vivo serum sterol concentration uponconsumption. Unfortunately, typical sterols and stanols are insoluble inthe micelli phase of the alimentary canal and have only limitedsolubility in oils and/or fats or water. Hence, free sterols or stanolsthemselves are not optimum candidates for use in typical pharmaceuticalor dietary dosage forms as cholesterol reducing agents.

U.S. Pat. No. 5,502,045 discloses the interesterification of stanolswith a fatty acid ester from an edible oil to produce a waxysterol-ester mixture with improved fat solubility characteristics.Specifically, this patent discloses the reaction of sitostanolinteresterified to an edible oil such as rapeseed oil specifically via abase catalyzed transesterification reaction. This is a process that iswidely used in the food industry. From a pharmaceutical standpoint,however, interesterification processes such as this have some distinctdisadvantages. Primarily, the composition profile of the sterol-esterproducts are difficult to control since the profile is dependent on thearray of fatty acids present in the edible oil employed in the reaction.

In a different approach, German Patent 2035069 discloses theesterification of sterol-esters to fatty acids via a non-food gradeprocess. In particular, thionyl chloride is employed as a reactant whichwhen reacted forms HCl gases as a by-product. Such techniques are notsuitable for the production of food grade materials, and they areundesirable in general for large scale reactions.

From a pharmaceutical standpoint, there is an unmet need for a methodfor the synthesis of discreet stanol/sterol-esters via a bulk food gradeprocess. Discreet compounds are more desirable than mixtures for threemain reasons: 1) the composition and performance specifications can becontrolled better; 2) structure/activity studies are more feasible; and3) the physicochemical and chemical properties can be controlled. Theseadvantages of discreet stanol/sterol-esters will be elaborated on later.

SUMMARY OF THE INVENTION

The present invention comprises a method for the direct esterificationof stanols or sterols with fatty acids to form discreetstanol/sterol-esters. The method provides a synthetic route that isamenable to large scale production of the stanol-esters in high yieldand purity by a food grade process that in a preferred embodiment isfree of organic solvents or mineral acids. The method ultimatelyprovides a convenient process that enables one to rationally designdiscreet stanol/sterol-esters with various physical and biologicalproperties.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the direct esterification of stanols andsterols through the reaction of the stanol/sterol and a fatty acid usinga food grade acid catalyst. β-sitostanol, the most preferred startingmaterial, is commercially produced from β-sitosterol by a hydrogenationreaction and is commercially available, from various sources includingHenkel Corporation.

The fatty acids reacted in the present invention are of the formulae CH₃--(CH₂)_(n) --CO₂ H wherein n is an integer of from 4 to 20. The termfatty acid is well known and understood to those with skill in the art,see for example, Hawley's Condensed Chemical Dictionary, Eleventhedition. The fatty acids include both saturated acids, such as stearic,butyric, lauric, palmitic and the like. Unsaturated fatty acids can alsobe used in the present invention and include oleic, linoleic, linolenic,docosohexanoic acid and the like.

In the present invention the sterol and stanol-esters have the generalformula depicted as FIG. I. ##STR1## wherein R is understood to includealiphatic straight or branched carbon chains ranging from C₆ -C₂₃,preferably from C₆ -C₂₀ and most preferably C₁₂ -C₁₈ groups.Unsaturation at C5 as shown gives the corresponding sterol-ester.

Any stanol or sterol that is functionalized with a hydroxy group issuitable for esterification by the process described herein. Stanolsthat are capable of being esterified in the present invention include,but are not limited to β-sitostanol and cholestanol, (depicted in FIG.II below). ##STR2## For example, this process is also amenable tosterols such as β-sitosterol (unsaturated at C5, as shown in FIG. Iabove).

The molar ratios of the starting materials for the esterificationreaction, notably the stanol/sterol and the fatty acid, are provided instoichiometric levels. In a highly preferred embodiment, the fatty acidis present in a 5-10% excess so as to react all of the stanol. Anyexcess unreacted fatty acid is easily removed in the product workup.

The acid catalyst is typically sufficient if provided at a 1 molepercent as compared to the reactants. The level of catalyst can beincreased or decreased to provide the reaction rate desired, however, iftoo much catalyst is provided a higher than desired level side productsmay result. Suitable acid catalysts include toluene sulfonic acid,methane sulfonic, sodium hydrogen phosphate, sodium bisulfate and thelike. Any acidic proton source can function as the catalyst, althoughstrong mineral acids are not preferred since their use may lead to somedecomposition of unsaturated fatty acids during the esterificationprocess. Sodium bisulfate is the preferred catalyst. The catalyst can bein the form of a solid, liquid or gas. Aqueous soluble catalysts arehighly preferred since they can easily be removed from the product withwater.

One of the most effective aspects of the present invention is that thereaction is performed neat, wherein no solvents are added to thereaction mixture, because the molten fatty acid acts as both a reactantand solvent.

It is particularly appropriate to run the neat reactions under vacuum inorder to remove water from the reaction mixture thereby driving thereaction to completion and increasing the yield of the desired ester.

The reaction temperature is conducted at temperatures from about 75° toabout 200° C. The preferred range is from about 100° to about 175° C.and most preferably from about 140° to 150° C. The reaction period mayvary widely, but for best results and economy the reactions should beallowed to run to completion. Reaction times of greater than 12 hoursare common but not necessarily required. One advantage of the presentinvention is the high yield of the ester product provided by theprocess. The present process provides yields of greater than 90% andpreferably greater than 95%.

Two isolation techniques as described below can be used to isolate theester reaction product.

Method A

An aqueous/organic solvent extraction isolation may be employed torecover the stanol-ester. Typical organic solvents includedichloromethane, chloroform or toluene. A typical aqueous/organic workupwas employed where the ester was extracted into an organic solvent andsubsequently isolated after evaporation. For example, the reactionmixture is cooled to room temperature followed by addition of CH₂ Cl₂.The solution was then washed several times with aqueous NaHCO₃. Thefatty acid salts are partitioned into the aqueous phase and can easilybe removed. The remaining organic phase containing the isolated ester isthen dried over anhydrous NaSO₄ and decolorized with activated charcoal.When light, non-chlorinated organic solvents (i.e., hexane) are used forextraction, the formation of an inseparable emulsion is observed. Pureesters were recovered as white solids or oils after removal of thesolvent on a rotary evaporator and subsequent cooling.

Method B

In a more preferred isolation technique, the ester reaction product isisolated using only water. The crude reaction mixture was diluted with1% aqueous NaHCO₃ and the resulting suspension was stirred rapidly for 1hour. The pure ester (>95% recovered yield) was filtered and vacuumdried overnight. A colormetric test for sulfate anion was performed on asmall sample of the ester, which showed that no catalyst remained amongthe product.

Although both methods produced esters identical in purity, the recoveredyields (>96%) were better with Method B. This method is also moreamenable to large scale synthesis because it gives high purity productwithout the use of hazardous non-food grade solvents.

The present invention provides several advantages over previousdisclosed processes. The present invention provides a method tosynthesize substantially discreet stanol-esters rather than mixtures ofstanol-esters. As used herein, substantially discreet is understood tomean that the reaction product, the desired ester is provided in a veryhigh proportion of the reaction product. Typically the desired ester isprovided in the reaction product in at least 90 percent by weight, morepreferably in an amount at least about 98 percent and if the reaction isallowed to run to completion to at least 99 percent by weight. Thepresent invention is capable of providing essentially a single stanol(sterol)-ester, with less than 0.2 weight percent of other esterproducts. The previously disclosed interesterification processes providea mixture of the stanol-ester products. For example, the previouslydisclosed processes provide mixtures of stanol-esters, often with broadranges of the stanol-esters present (for example, a mixture of 4 estersin ratios of 30, 30, 20, 20 percent by weight). Also in comparison, thepreviously disclosed direct esterification processes use hazardous,deleterious reagents.

This production of a discreet stanol/sterol-esters has several importantadvantages over the stanol/sterol-ester mixtures produced by otherprocesses. Firstly, tighter performance specifications (i.e., meltingpoint, specific gravity structural species purity) are possible fordiscreet compounds. This is because the properties of discreet compoundscan be controlled with more precision than for mixtures. Hence, properperformance characteristics and quality of discreet esters are moreeasily assured as compared to a mixture of ester products.

Furthermore, because the present invention provides the synthesis ofdiscreet stanol/sterol-esters, structure/activity relationships over arange of fatty acid chain lengths can be ascertained. The determinationof structure/activity relationships, which are fundamental to rationaldrug development, are only feasible when screening discreet compounds.

Finally, the gross physical and physiologic properties of thesterol/stanol-ester can be controlled since those properties aredependent upon which fatty acid is employed. For example, esterificationto unsaturated fatty acids (i.e., oleic acid) can lead to low meltingsolids or even liquid products, whereas saturated fatty acid analogs(i.e., stearic acid) tend to lead to higher melting free flowing solids.This ability to so extensively manipulate the physical properties of ahigh melting steroid is quite unexpected.

The present invention allows the selection of the ester to match thephysical properties which are desired. The solid free flowing materialis desirable for the manufacture of compressed tablets, or theincorporation of the stanol-ester into baking products. These oil-likestanol/sterol-esters are advantageously employed in the manufacture ofsoft gel dosage forms or incorporated into a salad dressing or yogurt.

The following examples are provided to further illustrate the claimedinvention, but not limit the invention to the examples provided below.

EXAMPLES

The stanol-fatty acid-esters of the invention were prepared by the acidcatalyzed esterification reaction method as follows: stanol (10 mmol),fatty acid (12 mmol) and sodium bisulfate (0.12 mmol) were stirred neatunder vacuum for 16 hours, at 150° C. The resulting stanol-esterproducts were isolated using either the technique described above asMethod A (employing both water and an organic solvent) or Method B (anaqueous separation process). When glass-like products were formed inmethod A, they were converted into free flowing solids upon coolingbelow 0° C. Gas chromatography analysis of crude reaction productindicated that the reactions proceed to greater than 95% completion.Final work-up was performed according to methods A or B as describedabove.

Analytical data for five representative stanol-esters are describedbelow. Analytical data for an ester of cholestanol, as an additionalmodel is also included.

Example 1

β-Sitostanol Stearate was produced by the reaction of β-sitostanol andstearic acid. NaHSO₄ was used as the catalyst and the stigmastanolstearate was isolated using Method A described above. The analyticalresults for the isolated stigmastanol stearate was as follows:

¹ HNMR (CDCl₃): (4.60(quintet, 1H), 2.19(t, 8, 2H), 1.88(d, 12, 1H); IR(cm⁻¹, KBr): 1739(s, C═O), 1454(m), 1388(m), 1182(s, C--O), 725(m);Elemental Analysis for C₄₇ H₈₆ O₂ : calculated: C 82.55% H 12.59%,found: C 82.70% H 12.50%; Melting Point (DSC): 103°-105° C.

Example 2

β-Sitostanol Stearate was produced by the reaction of β-sitostanol andstearic acid. NaHSO₄ was the catalyst used and the stigmastanol stearatewas isolated using Method B as described above. The analytical resultsof the isolated compound is presented below:

¹ HNMR (CDCl₃): (4.62, quintet, 1H), 2.18(t, 8, 2H), 1.88(d, 12, 1H); IR(cm⁻¹, KBr): 1739(s, C═O), 1467(m), 1381(m), 1176(s, C--O), 718(m);Elemental Analysis for C₄₇ H₈₆ O₂ : calculated: C 82.55% H 12.59%,found: C 82.31% H 12.63%; MP (DSC): 101°-104° C.; % H₂ O (Karl Fischer)0.73%

Example 3

β-Sitostanol Palmitate was produced by the reaction of β-sitostanol andpalmitic acid. NaHSO₄ was employed as a catalyst and the stigmastanolpalmitate was isolated using the procedure described above as Method A.The analytical results of the isolated stigmastanol palmitate ispresented below:

¹ HNMR (CDCl₃): (4.68(quintet, 1H), 2.24(t, 8, 2H), 1.95(d, 12, 1H); IR(cm⁻¹, KBr): 1739(s, C═O), 1460(m), 1394(m), 1176(s, C--O), 725(m);Elemental Analysis for C₄₅ H₈₂ O₂ : calculated: C 82.57% H 12.54%,found: C 82.59% H 12.53%; Melting Point (DSC): 102°-104° C.

Example 4

β-Sitostanol Oleate was produced by the reaction of β-sitostanol andoleic acid. NaHSO₄ was employed as a catalyst and the stigmastanololeate was isolated using the technique described as Method B. Theanalytical results are presented below:

¹ HNMR (CDCl₃): (5.27(m, 2H), 4.62(quintet, 1H), 2.23(t, 8, 2H); IR(cm⁻¹, neat): 1739(s, C═O), 1461(m), 1387(m), 1176(s, C--O), 1010(m),718(m); Elemental Analysis for C₄₇ H₈₄ O₂ : calculated: C 82.80% H12.33%, found: C 82.98% H 12.36%; Melting Point (DSC): 41°-44° C.

Example 5

Cholestanol Oleate was produced by the reaction of cholestanol and oleicacid. NaHSO₄ was used as a catalyst and the cholestanol oleate wasisolated using the technique described as Method A. The analyticalresults are presented below:

¹ HNMR (CDCl₃): (5.30(m, 2H), 4.65(quintet, 1H), 2.22(t, 8, 2H); IR(cm⁻¹, neat): 1725(s, C═O), 1454(s), 1367(m), 1168(m, C--O), 1003(m),711(m); Elemental Analysis for C45H80O2: calculated: C 82.67% H 12.25%;found: C 82.64% H 12.34%; Melting Point (DSC): 20°-25° C.

Comparative Example

The reaction of canola oil and stanol by an interesterification routeprovides a product mixture having the following approximate,non-reproducible distribution by weight:

Stanol-oleate 67%

Stanol-linoleate 19%

Stanol-linolenate 9%

Stanol-palmitate 3%.

I claim:
 1. A method for producing stanol/sterol esterscomprisingproviding a stanol/sterol of the formula ##STR3## providing afatty acid of the formula CH₃ --(CH₂)_(n) --CO₂ H wherein n is aninteger of from 4 to 20; reacting said stanol/sterol and fatty acid inthe presence of a mild acidic catalyst, resulting in the production ofthe substantially discrete corresponding stanol/sterol ester.
 2. Themethod of claim 1 wherein the reaction is conducted neat, with the fattyacid acting as the solvent.
 3. The method of claim 1 wherein the mildacid catalyst is NaHSO₄.
 4. The method of claim 1 wherein thecorresponding stanol/sterol ester is provided in an amount not less thanabout 98% by weight.
 5. The method of claim one wherein the reactiontemperature is from about 100° to about 200° C.
 6. The method of claimone wherein the reaction is run under vacuum.
 7. The method of claim onewherein the isolation of the corresponding stanol/sterol-ester isperformed in a completely aqueous process.